This is a continuing proposal for the study of the biochemical and molecular bases of ornithine (OTCase) deficiency and related inborn errors of urea genesis metabolism. Our previous studies have focused on understanding the correlation between OTCase structure and function and on elucidating the pathophysiology of its inherited defects. We now propose to expand this research to other systems of nitrogen metabolism to better understand their relationship to the urea cycle and its inherited defects. As components of urea genesis are distributed within both the mitochondria and cytosol, transporters of intermediates between the two compartments are required for efficient flux. The specific aims are to: (1) Study the structural and biochemical properties of human native and recombinant wild type mitochondrial ornithine carrier (ORNT) and mutants causing HHH syndrome (a) Study the oligomeric structure of ORNT by using hydrodynamic analysis, gel filtration, charge shift electrophoresis and cross-linking methods. (b) Investigate the topology and orientation of ORNT reconstituted into liposomes and in native mitoplasts and probe for domains critical for transport activity (c) Study several mutations that cause HHH syndrome to understand their deleterious effects on the function of ORNT (2) Investigate functional and structural interactions between ORNT and OTCase (a) Investigate structural interactions between OTCase and ORNT using co-precipitation, chemical cross-linking and protein foot-printing methods (b) Investigate functional interactions between the two proteins by fluorescence energy transfer and substrate interference experiments (c) Identify domains involved in interactions between ORNT and OTCase using site-directed mutagenesis. (3) Citrin (CITR) is a newly described calcium-binding aspartate/glutamate mitochondrial membrane carrier the absence of which, cause hyperammonemia and a form of citrullinemia (type II). We will characterize the function of CITR and investigate its interaction with argininosuccinate synthetase (AS) (a) Study the recombinant CITR in liposomes (b) Investigate the relationship and interactions between CITR and AS as well as VDAC (a mitochondrial porin protein) (4) We have recently cloned a novel mouse gene (NAGS/K) highly expressed in liver and intestine that has sequence similarity to yeast and bacterial N-acetylglutamate synthase (NAGS) and kinase (NAGK). We will characterize this gene (a) Study if the recombinant protein has NAGS or NAGK activity (b) Investigate the function of this gene by complementation studies in bacteria and yeast (c) Investigate mitochondrial targeting of this protein using Baculovirus expression in insects (d) Clone the mammalian NAGS gene by yeast complementation.